The Over Expression, Purification, and Characterization of a Protein in Bacteria
Introduction
In the three sessions this lab was carried out over, the plasmid pGEM-gbr22 which carries a gene for ampicillin resistance, has six histidine residue tags, and encodes for a fluorescent protein of origin from a coral in the Great Barrier Reef, was utilized. It’s fluorescent purple color allows for easy following of it’s expression and purification. It was over expressed in E. coli BL21, and purified by binding it’s histidine residue affinity tag to nickel in the Ni-NTA resin, subsequently washed, and released with imidazole, all through chromatography. As well, characterization was carried out through SDS-PAGE electrophoresis in the separation of proteins by the application of an electrical field across the protein gel sample. SDS is an anionic detergent that functions in denaturing proteins and providing a distance migration of the protein through the gel that is related to it’s mass. After the expression, purification, and characterization of the protein is completed, a single purified protein should be obtained. The significance of over expressing, purifying, and characterizing a protein in research, is that it has many uses in studying the structures and functions of human proteins, without the hassle or biological hazards of obtaining human tissue. When conducting an over expression of a protein, there are variants in the strains of E. coli, tags, selection markers, purification methods, and characterization methods chosen [2]. For this lab, E. coli BL21(DE3), an affinity tag of six histidine residues at the C-terminus, ampicillin as the selection marker, cell lysing and Ni-NTA affinity chromatography, and an SDS-PAGE gel were the variants chosen. However, if the plasmid carrying the gene for ampicillin resistance carried a gene for carbenicillin resistance instead, it would be a more stable selection marker [1].
Materials and Methods
Protein Expression
On day 1, 25ul of bacteria were placed into DNA and control transformation tubes. 1ul of plasmid was added to the DNA tube. The tubes were heat shocked in 42 degrees celsius for 45 seconds. 200ul SOC media was added, and they were shaken for 30min at 37 degrees celsius at about 250rpm. Bacteria was spread using colirollers. The fun plate was contaminated. The plates were covered, labeled, inverted, and stored in the 37 degrees celsius incubator overnight. On day 2, a single colony was picked, and put into a tube with LB/ampicillin, and left to grow in the shaking incubator at 37 celsius and 200-350rpm. 0.625ml of the started culture was transferred to flasks containing 25ml of fresh LB and ampicillin at 100ug/ml. Leave in shaking incubator at 37 celsius and 200-350rpm for 16-24hrs. On day 3, sample 1 was collected from the culture. The bacteria was spun down in a conical tube at 5,000rpm at 4 degrees celsius for 10mins. Purple pellet gathered at the bottom and the liquid was disposed of. The purple pellet was resuspended in PBS, vortex, and lysed.
Protein Purification
Throughout the purification, samples were collected for the characterization. The cells were lysed and clarified by centrifugation. The protein was purified by Ni-NTA affinity chromatography and stored at 4 degrees celsius.
Protein Characterization
Day 1, Through SDS-PAGE gel electrophoresis, the protein samples were prepared and ran through the gel for separation and migration of the proteins. The gel was stained for an hour using Imperial protein stain and then destained or rinsed. It was left in the shaker overnight. Day 2, the gel was dried using a vacuum bed.
ResultsPlate containing ampicillin and plasmid DNA pGEM-gbr22 seen as many purple colonies. One was chosen for the lab. Control Plate. Contains no plasmid DNA. No colonies observed. Ensured sterile/aseptic techniques. Fun plate containing a strand of mentor's hair. Contains no plasmid DNA. No growth observed. Large purple culture. Purple color verified the incubation growth time was complete and cells ready to harvest. Cell pellet obtained through centrifugation. MW of the pellet by tarring the conical tube was .68g for A and .56g for B. Elutions obtained from the protein purification. Elution 1 is a rich purple, while Elution 2 is a lighter purple. First reading obtained for Elution 1 using Protein A-280 setting. Absorbance at 280nm is 4.88 for 1cm path.Second reading obtained for Elution 1 using Protein A-280 setting. Absorbance at 280nm is 4.78 for 1 cm path.574nm being compared to 280nm, with 1.01 absorbance and .75 absorbance at 1cm path respectively. Beer's Law CalculationsC= A/(Eb) Molecular Weight of pGEM-gbr22 25794.2 g/molAt 280nm C= (.75)/((39100M^-1 cm^-1)(1cm))C= 1.98x10^-5 MMols= 1.98x10^-5M X .00005L = 9.9x10^-10 mols X 25794.2 g/mol = 2.55x10^-5g ProteinAt 574nmC= (1.01)/((118300M^-1 cm^-1)(1cm))C= 8.54x10^-6 MMols= 8.54x10^-6M X .0000L = 4.7x10^-10 mols X 25794.2 g/mol = 1.10x10^-5g Protein Ferments Page Ruler Prestained Protein Ladder #SM0671 is in Lanes 2 and 9. Samples 1-6 are in lanes 3-8 respectively. Lane 1 is empty. Lane 8 or Sample 6 should only show one protein band, but instead exhibits two.
Discussion
The protein purification lab was repeated after a mistake with the wash and elution in the first lab run. In the protein characterization lab, the protein migration was taking a long time, and after many attempts and retries to get it going, it was noted that the tape on the bottom of the gel was not removed prior to running the gel. The final dried gel shows two protein bands, where it should only be showing one protein band. This error could be brought about by many things, one being the storage of the Ni-NTA agarose column from the first attempt at the lab or the reuse of the Ni-NTA agarose column. As well, it could have been an error with the micropipetting of samples into the wells in the SDS-PAGE gel run, or the micropipettor itself. The estimated purity is about 50%. In sample 5, the estimated purity is about 33%.
Conclusions
The completion of this lab taught many techniques in an orientation of protein expression, purification, and characterization. Given the amount of errors, it seems that many of the techniques in this lab are a matter of practice and experience. After completing the expression, purification, and characterization of the protein, it was stored and may be used at a later time for another lab. The steps of this lab are all keystone steps, messing up early on would compromise the entire lab and results. However, running this lab again with extra care and precaution would definitely bring about different results than what was seen in the end product, SDS-PAGE gel.
Introduction
In the three sessions this lab was carried out over, the plasmid pGEM-gbr22 which carries a gene for ampicillin resistance, has six histidine residue tags, and encodes for a fluorescent protein of origin from a coral in the Great Barrier Reef, was utilized. It’s fluorescent purple color allows for easy following of it’s expression and purification. It was over expressed in E. coli BL21, and purified by binding it’s histidine residue affinity tag to nickel in the Ni-NTA resin, subsequently washed, and released with imidazole, all through chromatography. As well, characterization was carried out through SDS-PAGE electrophoresis in the separation of proteins by the application of an electrical field across the protein gel sample. SDS is an anionic detergent that functions in denaturing proteins and providing a distance migration of the protein through the gel that is related to it’s mass. After the expression, purification, and characterization of the protein is completed, a single purified protein should be obtained. The significance of over expressing, purifying, and characterizing a protein in research, is that it has many uses in studying the structures and functions of human proteins, without the hassle or biological hazards of obtaining human tissue. When conducting an over expression of a protein, there are variants in the strains of E. coli, tags, selection markers, purification methods, and characterization methods chosen [2]. For this lab, E. coli BL21(DE3), an affinity tag of six histidine residues at the C-terminus, ampicillin as the selection marker, cell lysing and Ni-NTA affinity chromatography, and an SDS-PAGE gel were the variants chosen. However, if the plasmid carrying the gene for ampicillin resistance carried a gene for carbenicillin resistance instead, it would be a more stable selection marker [1].
Materials and Methods
Protein Expression
On day 1, 25ul of bacteria were placed into DNA and control transformation tubes. 1ul of plasmid was added to the DNA tube. The tubes were heat shocked in 42 degrees celsius for 45 seconds. 200ul SOC media was added, and they were shaken for 30min at 37 degrees celsius at about 250rpm. Bacteria was spread using colirollers. The fun plate was contaminated. The plates were covered, labeled, inverted, and stored in the 37 degrees celsius incubator overnight. On day 2, a single colony was picked, and put into a tube with LB/ampicillin, and left to grow in the shaking incubator at 37 celsius and 200-350rpm. 0.625ml of the started culture was transferred to flasks containing 25ml of fresh LB and ampicillin at 100ug/ml. Leave in shaking incubator at 37 celsius and 200-350rpm for 16-24hrs. On day 3, sample 1 was collected from the culture. The bacteria was spun down in a conical tube at 5,000rpm at 4 degrees celsius for 10mins. Purple pellet gathered at the bottom and the liquid was disposed of. The purple pellet was resuspended in PBS, vortex, and lysed.
Protein Purification
Throughout the purification, samples were collected for the characterization. The cells were lysed and clarified by centrifugation. The protein was purified by Ni-NTA affinity chromatography and stored at 4 degrees celsius.
Protein Characterization
Day 1, Through SDS-PAGE gel electrophoresis, the protein samples were prepared and ran through the gel for separation and migration of the proteins. The gel was stained for an hour using Imperial protein stain and then destained or rinsed. It was left in the shaker overnight. Day 2, the gel was dried using a vacuum bed.
Results
Discussion
The protein purification lab was repeated after a mistake with the wash and elution in the first lab run. In the protein characterization lab, the protein migration was taking a long time, and after many attempts and retries to get it going, it was noted that the tape on the bottom of the gel was not removed prior to running the gel. The final dried gel shows two protein bands, where it should only be showing one protein band. This error could be brought about by many things, one being the storage of the Ni-NTA agarose column from the first attempt at the lab or the reuse of the Ni-NTA agarose column. As well, it could have been an error with the micropipetting of samples into the wells in the SDS-PAGE gel run, or the micropipettor itself. The estimated purity is about 50%. In sample 5, the estimated purity is about 33%.
Conclusions
The completion of this lab taught many techniques in an orientation of protein expression, purification, and characterization. Given the amount of errors, it seems that many of the techniques in this lab are a matter of practice and experience. After completing the expression, purification, and characterization of the protein, it was stored and may be used at a later time for another lab. The steps of this lab are all keystone steps, messing up early on would compromise the entire lab and results. However, running this lab again with extra care and precaution would definitely bring about different results than what was seen in the end product, SDS-PAGE gel.
References